No effect of creatine supplementation on oxidative stress and cardiovascular parameters in spontaneously hypertensive rats

Background Exacerbated oxidative stress is thought to be a mediator of arterial hypertension. It has been postulated that creatine (Cr) could act as an antioxidant agent preventing increased oxidative stress. The aim of this study was to investigate the effects of nine weeks of Cr or placebo supplementation on oxidative stress and cardiovascular parameters in spontaneously hypertensive rats (SHR). Findings Lipid hydroperoxidation, one important oxidative stress marker, remained unchanged in the coronary artery (Cr: 12.6 ± 1.5 vs. Pl: 12.2 ± 1.7 nmol·mg-1; p = 0.87), heart (Cr: 11.5 ± 1.8 vs. Pl: 14.6 ± 1.1 nmol·mg-1; p = 0.15), plasma (Cr: 67.7 ± 9.1 vs. Pl: 56.0 ± 3.2 nmol·mg-1; p = 0.19), plantaris (Cr: 10.0 ± 0.8 vs. Pl: 9.0 ± 0.8 nmol·mg-1; p = 0.40), and EDL muscle (Cr: 14.9 ± 1.4 vs. Pl: 17.2 ± 1.5 nmol·mg-1; p = 0.30). Additionally, Cr supplementation affected neither arterial blood pressure nor heart structure in SHR (p > 0.05). Conclusions Using a well-known experimental model of systemic arterial hypertension, this study did not confirm the possible therapeutic effects of Cr supplementation on oxidative stress and cardiovascular dysfunction associated with arterial hypertension.


Introduction
It has been suggested that exacerbated oxidative stress and its consequent oxidative damage may be mediators involved in cardiovascular diseases, such as systemic arterial hypertension [1]. Supporting this notion, a reduction in antioxidant bioavailability along with increased oxidative stress has been reported in both experimental and human hypertension [2].
Creatine (Cr) supplementation has emerged as a promising adjunct therapy in several pathological conditions [3], including cardiovascular diseases [4,5]. Interestingly, a growing body of experimental and clinical literature has suggested that Cr may exert protective effect in diseases where exacerbated oxidative stress plays a detrimental role (e.g., Huntington's disease) [6][7][8]. In fact, in vitro experiments have revealed that Cr may possess antioxidant properties by acting as a scavenger of free radicals, such as superoxide anions and peroxynitrite [8,9]. For instance, Cr pre-loading was found to be cytoprotective in different cell cultures with oxidative stressors (i.e., H2O2, tBOOH and peroxynitrite) [10]. Moreover, Cr may also "indirectly" attenuate the formation of reactive oxygen species trough the coupling of Cr with ATP into the mitochondria, ultimately resulting in a more efficient mitochondrial respiration and delayed accumulation of ADP f (i.e., the concentration of unbound ADP in the cytoplasm), which has been implicated in IMP and subsequently ROS formation [8,11]. This latter, in turn, may lead to oxidative stress with formation of chemical products of ROS reactions, such as oxidised glutathione and lipid hydroperoxides [12]. Despite the potential antioxidant capacity of Cr supplementation, its effects on oxidative stress and, consequently, cardiovascular parameters in experimental models of hypertension are still unknown. This is a short-report on the effects of Cr supplementation on oxidative stress, heart structure, and arterial blood pressure in spontaneously hypertensive rats (SHR), a well-established experimental model of arterial hypertension [13].

Procedures
This study was approved by the institution's ethical committee and was conducted in accordance with the National Research Council's Guidelines for the Care and Use of Laboratory Animals. Male SHR (26 weeks old) were housed under controlled environmental conditions (22°C; 12:12-h light:dark period) with free access to commercial chow and water. Animals were randomly allocated into two groups to receive either Cr (n = 8; 5 g/kg/d) or placebo (Pl; n = 7; distillated water). The groups have similar body mass (Cr = 324.7 ± 41.9 vs. Pl = 325.2 ± 21.6; p = 0.97). Cr monohydrate was administered by gavage for nine weeks. Forty-eight hours after the intervention, arterial blood pressure and heart rate were invasively measured using a catheter inserted into the femoral artery [14]. Thereafter, animals were killed by decapitation. Plasma, heart, carotid artery, plantaris, and extensor digitorum longus (EDL) muscles were isolated, weighted and deep frozen at -80°C for further analyses. Cardiomyocyte width and cardiac collagen deposition were also assessed by histological analyses, as measures of cardiac remodeling [15]. Additionally, lipid hydroperoxidation (an important marker of oxidative stress) was determined in the plasma, heart, carotid artery, and skeletal muscles. These aforementioned methods have been described in details below.

Hemodynamic parameters
After an intra-peritoneal anesthetic injection (80 mg/kg ketamine and 12 mg/kg xylazine, i.p.), a catheter filled with 0.06 mL of saline was inserted into the femoral artery of rats. Twenty four hours after the catheter insertion, the arterial cannula was connected to a straingauge transducer (Blood Pressure XDCR; Kent Scientific, Torrington, CT, USA), and arterial pressure signals were recorded over a 30 min period in conscious rats by a microcomputer equipped with an analog-to-digital converter board (WinDaq, 2 kHz, DATAQ, Springfield, OH, USA). The recorded data were analyzed on a beatto-beat basis to quantify systolic, diastolic and mean arterial pressure, as well as heart rate.

Histological analyses
Cardiac chambers were fixed by immersion in 4% buffered formalin and embedded in paraffin for routine histologic processing. Sections (4 μm) were stained with hematoxylin and eosin for examination by light microscopy. Only nucleated cardiac myocytes from areas of transversely cut muscle fibers were included in the analysis. Quantification of left ventricular fibrosis was achieved by Sirius red staining. Cardiac myocyte width and ventricular fibrosis were measured in the LV free wall with a computer assisted morphometric system (Leica Quantimet 500, Cambridge, UK).

Lipid hydroperoxidation measurement
Lipid hydroperoxidation was assessed since this oxidative stress marker has been implicated in the pathogenesis of a number of cardiovascular diseases, including arterial hypertension [16,17]. Lipid hydroperoxides were evaluated by the ferrous oxidation-xylenol orange technique (FOX2) [18]. Plasma, Heart, Carotid Artery, Plantar and EDL samples were homogenized in phosphate-buffered saline (PBS; 100 mmol/L, pH 7.4) and immediately centrifuged at 12.000 g for 20 min at 4°C. The homogenate was precipitated with trichloroacetic acid (10% w/v) and centrifuged (12.000 g for 20 min at 4°C). Supernatant was mixed with FOX reagent (250 mmol/L ammonium ferrous sulfate, 100 mmol/L xylenol orange, 25 mmol/L H2SO4 and 4 mmol/ L BHT in 90% methanol) and incubated at room temperature for 20 min. The absorbance of the sample was read at 560 nm in a spectrophotometer.

Statistical analysis
Data are expressed as mean ± standard error. The dependent variables were tested by unpaired Student's t test. Cohen's d effect size (Cr group minus placebo group divided by the standard deviation pooled) was also calculated for dependent variables. The level of significance was previously set at p < 0.05.

Results
As shown in Table 1, there were no significant differences in hemodynamic parameters between groups following the intervention.
Additionally, no significant differences between groups were shown in heart weight, cardiomyocyte width, and cardiac collagen content ( Table 2). Lipid hydroperoxidation also remained unchanged in the coronary artery, heart, plasma, plantaris, and EDL (Table 3).

Comments
Cr intake failed to attenuate oxidative stress in the cardiovascular system (i.e., heart and artery) as well in other tissues (i.e., plasma and skeletal muscle) in SHR. Furthermore, Cr did not affect either the heart structure or the hemodynamic parameters. Altogether, these data suggest that Cr supplementation does not exert therapeutically relevant effects in a model of SHR.
It has been speculated that the coupling of Cr with ATP into the mitochondria could attenuate the formation of reactive oxygen species by stimulating the respiration rate and reducing the free energy required for ATP synthesis [8]. Furthermore, Cr appears to act as a direct scavenger of radical species in face of oxidative stress [8,9]. These in vitro antioxidant proprieties confer to Cr a possible therapeutic role in diseases in which oxidative stress is exacerbated and related to pathological conditions. However, the current results were in contrast to our hypothesis. There are two potential speculations for the lack of any "positive" outcome in this study. First, the arterial blood pressure peaks at 24 weeks of age in SHR [13]. Therefore, one may assumedespite the lack of a healthy control group -that our rats displayed severe arterial hypertension. In such extreme conditions, Cr may be not capable of reverting cardiovascular dysfunction. Second, Cr metabolism is divergent among species [19], meaning that the in vitro antioxidant effects of Cr may not be extended to in vivo models. Further studies with other experimental models of hypertension as well as randomized controlled trials with humans are required to determine whether Cr supplementation can alleviate oxidative stress and cardiovascular dysfunction in arterial hypertension.
In summary, Cr supplementation did not affect oxidative stress or cardiovascular parameters in SHR model.